Fig. 2: Crystal structures of HG-series Kemp eliminases.

In all cases, only atoms from chain A are shown. a Binding pose of the 6-nitrobenzotriazole (6NT) transition-state analogue (orange). Hydrogen bonds are shown as dashed lines. The red sphere represents a water molecule. The 2Fo-Fc map is shown in volume representation at two contour levels: 0.5 and 1.5 eÅ⁻³ in light and dark blue, respectively. b 6NT (orange) is sandwiched between the hydrophobic side chains of Trp44 and Met237. c The peptide bond between residues 83 and 84 can adopt cis or trans conformations. Hydrogen bonds are shown as dashed lines. The 2Fo-Fc map is shown in volume representation at two contour levels: 0.5 and 1.5 eÅ⁻³ in light and dark blue, respectively. d Conformational changes to the loop formed by residues 87–90 over the course of the evolutionary trajectory. The 2Fo-Fc map is shown in volume representation at two contour levels: 0.5 and 1.5 eÅ⁻³ in light and dark blue, respectively. e Superposition of the 6NT-bound structure (white) with the highest (magenta) and lowest (green) occupancy conformers of the unbound structure for each Kemp eliminase. From HG3 to HG3.14, the unbound state is never pre-organized for catalysis as both Trp44 and Met237 adopt conformations that would prevent the productive binding of the transition state. In HG3.17 and HG4 however, only Trp44 adopts a non-productive conformation in the unbound state, with an occupancy of 62% or 26%, respectively. f Cut-away view of the active site shows that its entrance (top) becomes widened during evolution, as indicated by an increasing bottleneck radius (reported as the average radius ± s.d. calculated using the highest occupancy conformers from both chain A and B, except for HG3.17, which contains a single chain). 6NT is shown as orange spheres. Bottleneck radii were calculated using the PyMOL plugin Caver 3.0²².